Combined proteomic strategies for in-depth venomic analysis of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand.

This study focuses on comprehensive characterization of the venom proteome of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand. H. schistosus can be considered as the deadliest sea snake commonly found in the Pacific and Indian oceans. Their envenomation causes muscular paralysis and rhabdomyolysis. To develop effective treatment for this snakebite, it is necessary to understand the detailed venom composition. In this study, multiple mass spectrometry-based approaches were employed. Bottom-up proteomics revealed that tryptic digestion in-solution provided a higher number of toxin proteins identified and a larger sequence coverage, compared to in-gel digestion. In addition, a venom gland transcriptome-derived database was constructed and used as a reference, which 43 known and novel toxin proteins were identified using this database and the UniProtKB. Three-finger toxin and phospholipase A2 were shown to be top two most abundant protein families. Minor compositions included other toxin families and a number of non-toxin proteins. Moreover, a hybrid de novo sequencing was performed to enhance identification of the small proteins/peptides. Using non-digested samples, there were 46 predicted toxin peptides. The finding from this study could lead to a better understanding in pathological effects of the snakebite and the future development of effective antivenoms. SIGNIFICANCE: This study provides a better understanding of the venom proteome composition of the beaked sea snake (H. schistosus) found in the Gulf of Thailand, using a combination of different sample preparation techniques, Serpentes protein database searching, transcriptome-derived protein database searching, and a hybrid de novo peptide sequencing strategy. It revealed 13 toxin protein families and novel proteins in the beaked sea snake venom including new species of phospholipase A2s (PLA2s) and three-finger toxins (3FTxs). It could serve as a basis for the development of snakebite treatments and for the discovery of novel pharmaceutical drugs from the toxin peptides.

Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus.

Vector control, the most efficient tool to reduce mosquito-borne disease transmission, has been compromised by the rise of insecticide resistance. Recent studies suggest the potential of mosquito-associated microbiota as a source for new biocontrol agents or new insecticidal chemotypes. In this study, we identified a strain of Serratia marcescens that has larvicidal activity against Anopheles dirus, an important malaria vector in Southeast Asia. This bacterium secretes heat-labile larvicidal macromolecules when cultured under static condition at 25°C but not 37°C. Two major protein bands of approximately 55 kDa and 110 kDa were present in spent medium cultured at 25°C but not at 37°C. The Liquid Chromatography-Mass Spectrometry (LC-MS) analyses of these two protein bands identified several proteases and chitinases that were previously reported for insecticidal properties against agricultural insect pests. The treatment with protease and chitinase inhibitors led to a reduction in larvicidal activity, confirming that these two groups of enzymes are responsible for the macromolecule's toxicity. Taken together, our results suggest a potential use of these enzymes in the development of larvicidal agents against Anopheles mosquitoes.

Application of Higher Density Iron Oxide Nanoparticle Pellicles to Enrich the Plasma Membrane and Its Proteome from Cells in Suspension.

Enrichment of the plasma membrane represents one valuable method to characterize the surfaceome, along with other plasma membrane and structural proteins. Currently, the overlapping densities of many subcellular organelles hinder enrichment of the plasma membrane by centrifugation. However, external access to the plasma membrane of intact cells allows the attachment of a nanoparticle pellicle to enhance its density and facilitate enrichment. We describe the synthesis of iron oxide nanoparticles, attachment of the pellicle to suspended cells, and recovery of plasma membrane proteins for proteomic analysis.

Peptide-based systems analysis of inflammation induced myeloid-derived suppressor cells reveals diverse signaling pathways.

A better understanding of molecular signaling between myeloid-derived suppressor cells (MDSC), tumor cells, T-cells, and inflammatory mediators is expected to contribute to more effective cancer immunotherapies. We focus on plasma membrane associated proteins, which are critical in signaling and intercellular communication, and investigate changes in their abundance in MDSC of tumor-bearing mice subject to heightened versus basal inflammatory conditions. Using spectral counting, we observed statistically significant differential abundances for 35 proteins associated with the plasma membrane, most notably the pro-inflammatory proteins S100A8 and S100A9 which induce MDSC and promote their migration. We also tested whether the peptides associated with canonical pathways showed a statistically significant increase or decrease subject to heightened versus basal inflammatory conditions. Collectively, these studies used bottom-up proteomic analysis to identify plasma membrane associated pro-inflammatory molecules and pathways that drive MDSC accumulation, migration, and suppressive potency.

Nanowire pellicles for eukaryotic cells: nanowire coating and interaction with cells.

AIM: To construct a new robust nanowire-based pellicle for eukaryotic cells, to investigate the interactions between nanowires (NWs) and cell surfaces and the internalization of nanowires, and to demonstrate for isolation of the plasma membrane with improved enrichment of transmembrane proteins. MATERIALS & METHODS: Silica NWs were coated with alumina to give positive charges on their surface. Multiple myeloma cells were coated with the positively charged NWs by dropping the cells into a buffered suspension of NWs. After the NW-coated cells were lysed, plasma membrane fragments were enriched by differential centrifugation for proteomic studies. RESULTS: Here we demonstrate complete cell coating with positively charged, alumina-coated silica NWs via nonspecific electrostatic interactions, and characterize a robust pellicle and little/no uptake of NWs. CONCLUSION: Robust pellicles provide a new platform for therapeutic, diagnostic and biochemical interactions of nanostructures with eukaryotic cells.

Exosomes from myeloid-derived suppressor cells carry biologically active proteins.

Myeloid-derived suppressor cells (MDSC) are present in most cancer patients where they inhibit natural anti-tumor immunity and are an obstacle to anti-cancer immunotherapies. They mediate immune suppression through their production of proteins and soluble mediators that prevent the activation of tumor-reactive T lymphyocytes, polarize macrophages toward a tumor-promoting phenotype, and facilitate angiogenesis. The accumulation and suppressive potency of MDSC is regulated by inflammation within the tumor microenvironment. Recently exosomes have been proposed to act as intercellular communicators, carrying active proteins and other molecules between sender cells and receiver cells. In this report we describe the proteome of exosomes shed by MDSC induced in BALB/c mice by the 4T1 mammary carcinoma. Using bottom-up proteomics, we have identified 412 proteins. Spectral counting identified 63 proteins whose abundance was altered >2-fold in the inflammatory environment. The pro-inflammatory proteins S100A8 and S100A9, previously shown to be secreted by MDSC and to be chemotactic for MDSC, are abundant in MDSC-derived exosomes. Bioassays reveal that MDSC-derived exosomes polarize macrophages toward a tumor-promoting type 2 phenotype, in addition to possessing S100A8/A9 chemotactic activity. These results suggest that some of the tumor-promoting functions of MDSC are implemented by MDSC-shed exosomes.

Enrichment of plasma membrane proteins using nanoparticle pellicles: comparison between silica and higher density nanoparticles.

Proteomic and other characterization of plasma membrane proteins is made difficult by their low abundance, hydrophobicity, frequent carboxylation, and dynamic population. We and others have proposed that underrepresentation in LC-MS/MS analysis can be partially compensated by enriching the plasma membrane and its proteins using cationic nanoparticle pellicles. The nanoparticles increase the density of plasma membrane sheets and thus enhance separation by centrifugation from other lysed cellular components. Herein, we test the hypothesis that the use of nanoparticles with increased densities can provide enhanced enrichment of plasma membrane proteins for proteomic analysis. Multiple myeloma cells were grown and coated in suspension with three different pellicles of three different densities and both pellicle coated and uncoated suspensions analyzed by high-throughput LC-MS/MS. Enrichment was evaluated by the total number and the spectral counts of identified plasma membrane proteins.

Comparison of nanowire pellicles for plasma membrane enrichment: coating nanowires on cell.

A study is reported on the effect of nanowire density on the ease of pellicle formation and the enrichment of plasma membrane proteins for analysis by mass spectrometry. An optimized synthesis is reported for iron silicate nanowires with a narrow size range of 900 ±400 nm in length and 200 nm diameter. The nanowires were coated with Al2O3 and used to form pellicles around suspended multiple myeloma cells, which acted as a model for cells recovered from tissue samples. Lighter alumina-coated silica nanowires were also synthesized (Kim et al. 2013), which allowed a comparison of the construction of the two pellicles and of the effect of nanowire density on plasma membrane enrichment. Evidence is offered that the dense nanowire pellicle does not crush or distort these mammalian cells. Finally, the pellicles were incorporated into a mass-spectrometry-based proteomic workflow to analyze transmembrane proteins in the plasma membrane. In contrast to a prior comparison of the effect of density with nanoparticles pellicles (Choksawangkarn et al. 2013), nanowire density was not found to significantly affect the enrichment of the plasma membrane. However, nanowires with a favorable aspect for pellicle formation are more easily and reliably produced with iron silicate than with silica. Additionally, the method for pellicle formation was optimized through the use of iron silicate nanowires (ISNW), which is crucial to the improvement of PM protein enrichment and analysis.

Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins.

Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.